The objectives of this application are to use cellular models of mtDNA-linked diseases, constructed by mitochondria-mediated transformation of mtDNA-less (r[unreadable]) cells, in order 1) to determine in a definitive way the pathogenic mechanism at the molecular level of the most commonly occurring mitochondrial tRNA mutations responsible for the MERRF and MELAS encephalomyopathies; 2) to elucidate the factors that underlie the extraordinary tissue specificity associated with these pathogenic mtDNA mutations and the complex complementation and segregation behavior of these mutations; and 3) to understand the role of the human and mouse homologues of the Drosophila fzo protein controlling mitochondrial fusion and of the dynamin-related protein Drpl controlling mitochondrial fission in the processes of transcomplementation and segregation of mtDNA mutations. Specifically, the present proposal aims at determining the role of the nuclear background, as distinct from the mtDNA haplotype, in modulating the biochemical phenotype of mtDNA mutations responsible for the MERRF and MELAS encephalomyopathies in several r[unreadable] cell lines and in rhabdomyosarcoma cells differentiated in vitro into myotubes and in neuroblastoma cells differentiated into neuronal cells. Attention will be paid also to the role of the nuclear background in the complementation and segregation behavior of the tRNA mutations. It is also planned to study the effects of hyperexpression of the gene for Mfnl or Mfn2, human homologues of fzo, co-expressed with the gene for a dominant mutant form of Drpl, or the effects in mouse embryonic fibroblasts of the knock-out of the Mfzol or Mfzo2 gene, mouse homologues of fzo, in the transcomplementation and segregation of sequentially introduced mutant and wild-type mitochondrial genes. It is also planned to investigate the role of lowered 02 concentrations, comparable to the in vivo concentrations, in the differentiation of rhabdomyosarcoma and neuroblastoma cells and in the biochemical phenotype and complementation and segregation behavior of pathogenic mtDNA mutations in these differentiated cells. The achievement of the above aims will have significant implications for understanding the in vivo pathogenic mechanisms of mtDNA mutations causing diseases in man, their establishment and transmission. Furthermore, it is expected that this work will provide important insights into the regulation of mitochondrial gene expression and into fundamental features of mitochondrial genetics in mammalian cells.